Thrombin is postulated to mediate vascular lesion formation (VLF) at sites of denuding vascular injury. We have measured the effects of hirudin, a potent direct antithrombin, on VLF developing after surgical implantation of angioaccess vascular grafts (AVGs) in baboons. To avoid bleeding caused by chronic systemic infusions, hirudin was delivered locally into the vascular flow boundary layer by hirudin-transduced vascular endothelial cells (ECs) covering AVG luminal surfaces. cDNAs encoding hirudin were transferred to ECs by retroviral transduction in culture. Two EC-lined AVGs were surgically implanted into each of 7 baboons, one AVG lined with hirudin-control ECs, and the contralateral AVG lined with hirudin-secreting ECs. After 30 days both AVGs were harvested for EC recovery, histochemical and morphometric analysis of VLF on the graft surfaces and at both proximal and distal vessel-graft anastomoses. Hirudin-transduced ECs secreted gene product in vitro, i.e., 20q6 ng/1 06ECs/ 24 hr. Hirudin-secreting ECs also reduced thrombosis on segments of collagen-coated graft interposed in exteriorized AV shunts (111In-platelet deposition from 0.82q0.49 x109 platelets to 0.52q0.34 x109 platelets; p=0.03). Luminal ECs covering flow surfaces of the implanted AVGs remained attached and secreted hirudin (17q7 ng/106ECs/24 hr) after recovering EC from harvested AVGs. AVGs lined with hirudin-secreting ECs showed reduced VLF at distal graft-vessel anastomoses, i.e., 1.02 q 0.14 mm2 vs 1.82 q 0.15 mm2 in AVGs bearing non-hirudin control ECs (p <0.01). By contrast, upstream proximal anastomoses not perfused by hirudin showed no reduction in VLF, i.e., 1.89 q 0.22 mm2 vs 1.99 q 0.26 mm2 (p>0.5). Continuous inactivation of thrombin by hirudin at sites of AVG-vessel anastomoses significantly reduces neotintimal lesion formation, indicating the importance of thrombin in the generation of VLF. Because mononuclear leukocytes, but not neutrophils, depend on 4 (CD49d) integrin receptors for adhesion and activation, we investigated the effects of inhibiting mononuclear leukocyte attachment by injecting sufficient anti- 4 monoclonal antibody (anti- 4 MoAbs) to maintain saturating levels in the circulation. Bilateral surgical carotid endarterectomies were performed in 10 adult male baboons whose platelets had previously been labeled with 111In-oxine. Five animals received intraoperative intravenous injections of anti- 4 MoAbs (3 mg/kg), and a second injection two weeks later; the remaining five received saline control injections. Intravenous bolus injections of anti- 4 MoAbs produced circulating levels of antibody exceeding the saturating level of 0.1 fg/mL throughout the four post-operative weeks that induced elevated peripheral monocyte counts, and exhibited persistent inhibition of monocyte adhesion, ex vivo, despite the appearance of non-neutralizing anti-idiotypic antibodies directed against anti- 4 MoAbs during the second week. 111In-platelet deposition at sites of endarterectomy was not significantly changed by anti- 4 MoAb therapy, compared with untreated controls (p>0.2), thereby documenting the lack of antithrombotic activity for anti- 4 MoAbs. Neointimal vascular lesion formation (NVLF) at sites of endarterectomy was reduced to 0.33q0.05 mm2 (n=9) by treatment with anti- 4 MoAbs, compared with 0.55q0.15 mm2 (n=10) in the saline control cohort (p=0.01). The frequency of proliferating neointimal cells, as measured by proliferating cell nuclear antigen (PCNA)-positive cells, was decreased to 2.88q1.07 in treated animals, compared with 4.89q0.77% in untreated controls (p=0.001). The accumulation of macrophages in the neointimal proliferative lesions was reduced to 15.8q3.1 macrophages/section in the treated arteries, compared with 33.5q6.1 macrophages/section in untreated controls (p= 0.001). This study demonstrates that interruption of mononuclear adhe sion by anti- 4 MoAbs in endarterectomized baboons significantly reduces a) the accumulation of neointimal macrophages; b) the proliferation of neointimal cells; and c) the amount of neointimal vascular lesion formation, without decreasing vascular thrombosis, and identifies this approach as a possible strategy for reducing clinical restenosis. [unreadable] P51RR00165-36 1/1/96 - 12/31/96 Yerkes Regional Primate Research Center